In imaging systems, particularly microscopy, adequate and appropriate illumination of the object to be imaged is essential. There must be enough light provided to the object or specimen to be viewed to permit the viewer to discern features of the object. In addition, the manner in which the light is provided to the object makes a difference in what features can be detected and the contrast with which they are imaged.
An ordinary microscope typically employs a compound imaging lens system for imaging the object. Any number of lenses or other optical elements such as polarizers, collimators, spreading optics, mirrors, and splitters may be included in the lens system. The lens system may be characterized in part by its numerical aperture, which essentially defines the limiting angle at which light from the object can pass into the lens system.
The object to be imaged by a microscope is typically located at the object plane by being placed on a substrate that is, in turn, positioned on a stage of the microscope that can be moved laterally with respect to the optical axis of the lens system. The stage may be motorized so that this movement may be automated or controlled by a computer. Moreover, the image plane may be provided with a camera or other imaging device for recording the image, or for monitoring the image under the same computer control.
In addition to being characterized by its numerical aperture, an imaging lens system is also characterized by its field of view. The field of view in visible light microscopes typically ranges from tens of microns to a few millimeters. This means that a macroscopically sized object of, for example, 20 mm×50 mm requires many movements of the stage for imaging the entire object. The stage manipulation and the consequent time required to image an object under high magnification is particularly troublesome in pathology analysis since the diagnostic information in the tissue may be located in only a small portion of the object that is being imaged.
A recent innovation in the field of light microscopy that addresses this problem is a miniaturized microscope array (“MMA”) which, when applied to a common object, is also referred to as an “array microscope.” In miniaturized microscope arrays, a plurality of imaging lens systems are provided having respective optical axes that are spaced apart from one another. Each imaging lens system images a respective portion of the object.
In an array microscope, a linear array is preferably provided for imaging across a first dimension of the object, and the object is translated past the fields of view of the individual imaging elements in the array, so that the array is caused to scan the object across a second dimension to image the entire object. The relatively large individual imaging elements of the imaging array are staggered in the direction of scanning so that their relatively small fields of view are contiguous over the first dimension. The provision of the linear detector arrays eliminates the requirement for mechanical scanning along the first dimension, providing a highly advantageous increase in imaging speed.
As mentioned, microscopy depends on having an adequate source of light to illuminate the object. Several different types of illumination systems can be employed. One type illumination system is known as “epi-illumination.” In epi-illumination, light illuminates the surface of the object and is either reflected from or back-scattered by the object into the imaging lens system. This light may also be effectively transformed in wavelength by the object, as a result of the object's fluorescence, which is known as “epi-fluorescence.” Epi-illumination is necessary where the object to be imaged is opaque.
On the other hand, if the object to be imaged is not opaque, it can be illuminated by light transmitted through the object. This type of illumination is known as “dial illumination,” “through illumination,” or, as referred to herein, “trans-illumination.” An otherwise opaque object can be made to be light transmissive by cutting it into thin sections, or the object may be formed of transparent or partially transparent materials, such as biological materials. For example, pathologists routinely view tissue specimens and liquid specimens such as urine and blood using trans-illumination in a light microscope.
Trans-illumination typically makes use of an illumination lens system that projects light from a light source through the aforementioned substrate, through the object, and into the imaging lens system. The substrate is typically a glass or other transparent material slide, about 1 to 1.5 mm thick. The object to be viewed is mounted to or disposed on a front side of the substrate and light is applied to the object through the back side of the substrate. Since it is also formed of optical elements, the illumination lens system is governed by the same optical principles as the imaging lens system. Thence, the illumination lens system is likewise characterized by its numerical aperture and its field of view.
There are two primary trans-illumination systems. In one of these, known as Kohler illumination, the image plane of the illumination system is placed at the pupil plane of the imaging system, or a conjugate thereof, so that the light source is imaged into the pupil of the imaging system. The other primary illumination strategy is known as critical illumination. In this case, the image plane of the illumination system is placed at the object plane of the imaging system, or a conjugate thereof, so that the light source is imaged into the object plane of the imaging system. An advantage of Kohler illumination is that each point on the image plane experiences the average light intensity that is provided by the source, so that illumination is insensitive to spatial variations in source radiance. Critical illumination permits the optical system to be made shorter than that of a Kohler illumination system and increases illumination efficiency; however, it requires that the light source provide spatially uniform radiance.
The MMA concept invites the corresponding concept of providing each imaging element with a corresponding illumination element. For optimal effect, the numerical aperture of illumination lens systems need to be matched to the numerical aperture of their corresponding imaging elements. That is, if the illumination system transmits light to the object at angles greater than the acceptance angle of the imaging system, some of the light may be wasted, which reduces system efficiency. On the other hand, if the illumination system transmits light over a narrower angular range, that is, one that does not extend to the acceptance angle, the imaging system cannot take full advantage of its resolving power.
In a high numerical aperture array microscope it is desirable to pack the imaging elements of the array close together so as to acquire image data for contiguous parts of the object in the minimum scan time. On the other hand, a trans-illumination system places a limit on how close the corresponding illumination lens systems can be packed and still provide the desired matching of numerical apertures. This is because the object must be supported by a slide or other transparent member that must be sufficiently thick to provide mechanical stability. Where the illumination system must project light through a glass substrate 1 to 1.5 mm thick, the working distance cannot be greater than that amount. To have a sufficiently long illumination system working distance, while maintaining the same numerical aperture as the imaging system, the diameter of the lens of the illumination system must be larger than the diameter of the lens of the imaging element. This means that providing each imaging element with its own illumination element requires either that suboptimal imaging element packing or suboptimal numerical aperture matching must be employed.
Accordingly, there is an unfulfilled need for devices and methods for providing trans-illumination for arrays of imaging elements having respective optical axis, particularly array microscopes, without sacrificing either image element packing density or optimal numerical aperture matching.